This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recently, an approach pioneered in Prof. Peter Schultz lab (Scripps) allows for the site-specific incorporation of an unnatural amino acid anywhere within a protein sequence. This technology has already proven useful for labeling proteins with fluorescent, 13C and 15N, and chemical crosslinking unnatural amino acids for in vivo and in vitro structure-function studies. By using an orthogonal M. jannaschii tRNA/tRNA-synthetase pair, that is one where neither the tRNA nor the synthetase cross-reacts with the endogenous E.coli tRNA's or amino acyl tRNA sythetases, it is possible to introduce an unnatural amino acid residue at any position in the protein using a codon that is only recognized by the orthogonal tRNA. Through collaboration with the Schultz lab at Scripps, the Paul Ortiz de Montellano lab has been able to acquire this labeling technology in house and has recently used it to successfully label several heme containing proteins in preparation for further biophysical studies. Unfortunately, lack of information as to the extent of incorporation of the unnatural amino acid and relative expression levels has limited the application of this technology. To that end, we hope to utilize the UCSF Department of Pharmaceutical Chemistry Mass Spectrometry Facility to access the extent and location of incorporation of the unnatural amino acids into various proteins, and also determine their relative expression levels. This knowledge will not only further our work, but will also allow for more widespread adoption of this powerful technology.